Pulp regeneration compositions and methods of forming and using the same

ABSTRACT

A dental tissue regenerative composition. The composition includes a combination of (1) human dental pulp stem cells and (2) at least one of human umbilical vein endothelial cells or vascular endothelial growth factor. The combination is encapsulated in a light-activated gelatin methacrylate hydrogel.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 62/428,870, filed Dec. 1, 2016, which is herebyincorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.R01DE016132(PCY), awarded by the National Institutes of Health, theNational Institute of Dental and Craniofacial Research, and the NationalInstitute of Biomedical Imaging and Bioengineering. The government hascertain rights in the invention.

TECHNICAL FIELD

The present invention relates to compositions and methods for dentalpulpal revascularization, replacing infected dental pulpal tissues inpulpotomy, or temporary intra canal dental medicaments. Morespecifically, the invention relates to compositions including humandental pulp stem cells, human umbilical vein endothelial cells, and/orvascular endothelial growth factor encapsulated in a light-activatedgelatin methacrylate hydrogel and methods of forming and using the same.

BACKGROUND

Pulpal revascularization therapy is commonly used in dental clinics toobtain apical closure of immature permanent teeth with thin dentinalwalls and/or on injured teeth to promote continued root development andto prevent fracture of thin dentinal walls. Goals of this therapyinclude achieving apical closure development similar to that of adjacentteeth, preventing tooth and supporting bone loss, and/or minimizing theneed for and the financial burden associated with dental implantplacement.

Regardless of these good intentions, undesirable outcomes can occurfollowing clinical revascularization procedures. For example,stimulating bleeding from the periapical area of a tooth maydeleteriously affect tooth root maturation. Other undesirable outcomesthat may occur include arrested tooth root development, incompleteclosure of the tooth apex, calcification within the pulpal space thatmay impede future root canal treatment, combinations thereof, or thelike.

Induced bleeding at the tooth apex is a procedure used to activate theproliferation and migration of Stem Cells from Apical Papilla (SCAP)into the pulpal space and to release growth factors such asPlatelet-Derived Growth Factor (PDGF), which participate inangiogenesis. Unfortunately, insufficient bleeding is commonly observed,leading to possible arrested tooth root development.

Several in vivo studies have attempted to regenerate the dentin-pulpcomplex by incorporating cells such as human Dental Pulp Stem Cells(hDPSCs), Human Umbilical Vein Endothelial Cells (HUVECs), and SCAP.These in vivo studies utilized scaffolds such as PURAMATRIX (3-D Matrix,Ltd, Tokyo, Japan), nanofibrous gelatin/silica bioactive glass(NF-gelatin/SBG) hybrid, Collagen, poly L lactic acid (PLLA), andFlouroapetite crystal coated with poly caprolactone. Others studies havedemonstrated the ability to regenerate pulp-like tissue usingscaffold-free approaches, including cell sheath technology and DSCaggregates formed on agarose dishes.

3D biomimetic tooth bud models have been created usingphotopolymerizable Gelatin Methacrylate (GelMA) hydrogel. These modelwere designed to facilitate dental epithelial (DE) and dentalmesenchymal (DM) cell interactions leading to ameloblast and odontoblastdifferentiation, respectively, and the formation of bioengineered teethof predictable size and shape.

GelMA hydrogels exhibit numerous properties that make them useful for avariety of tissue engineering applications. For example, GelMA islargely composed of denatured collagen and retains many of collagen'snatural properties including RGD adhesive domains and MMP sensitivesites that enhance cell binding and cell-mediated matrix degradation. Inaddition, the physical properties of GelMA hydrogels can generally betuned by varying GelMA and/or photo-initiator (PI) concentrations. GelMAis also suitable for cell encapsulation at about 37° C. and forpromoting cell viability and proliferation. Further still, GelMA isrelatively inexpensive.

GelMA formulas exhibiting elastic moduli similar to natural dentalenamel and pulp organ tissues, respectively, have been identified. Suchformulas have been found to be suitable for bioengineered toothapplications. Incorporating HUVECs along with DE and DM cells inbioengineered 3D GelMA tooth bud constructs was found to promoteneovascular formation and facilitate in vivo engraftment with hostvasculature.

hDPSCs have recently been considered for use in pulpal regeneration.DPSCs have been found to exhibit the potential to regenerate dentin-pulpcomplex after being seeded onto poly-D,L-lactide/glycolide scaffold andtransplanted in vivo for about 3-4 months. However, existing challengesfor this approach include identifying a scaffold that truly mimics theECM of natural pulp and creating a sufficient blood supply to ensure thesurvival of in vivo transplanted DPSCs.

The below-described devices, methods, and systems address many of thesedeficiencies by using GelMA hydrogels and, specifically, GelMAencapsulated hDPSCs and HUVECs and/or vascular endothelial growth factor(VEGF) for clinically relevant applications for pulpal regeneration.

SUMMARY

According to aspects of the present disclosure, a dental tissueregenerative composition comprises a combination of (1) human dentalpulp stem cells and (2) at least one of human umbilical vein endothelialcells or vascular endothelial growth factor. The combination isencapsulated in a light-activated gelatin methacrylate hydrogel. It iscontemplated that other hydrogel scaffolds may also be used.

According to additional aspects of the present disclosure, a method ofregenerating pulp-like tissues in a tooth root segment of a toothincludes injecting a composition into the tooth root segment. Thecomposition includes gelatin methacrylate hydrogel and aphoto-initiator. The method further includes combining the compositionwith a mixture of (1) human dental pulp stem cells and (2) at least oneof human umbilical vein endothelial cells or vascular endothelial growthfactor. The method further includes cross-linking the gelatinmethacrylate hydrogel with the mixture by exposing the composition to aUV or visible light.

According to a further aspect of the present disclosure, a method offorming a dental tissue regenerative composition includes combininghuman dental pulp stem cells, at least one of human umbilical veinendothelial cells or vascular endothelial growth factor, a gelatinmethacrylate hydrogel, and a photo-initiator. The method furtherincludes cross-linking the gelatin methacrylate hydrogel with the humandental pulp stem cells and the at least one of human umbilical veinendothelial cells or vascular endothelial growth factor by exposure to aUV or visible light.

These and other capabilities of the inventions, along with theinventions themselves, will be more fully understood after a review ofthe following figures, detailed description, and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the invention is not intended to belimited to the particular forms disclosed. Rather, the invention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention as defined by the appended claims.

FIG. 1A is a schematic diagram showing: GelMA-encapsulated cellconstructs (G1) injected into human root segment (RS) andphotocrosslinked for about 30 seconds; acellular GelMA RSs (G2)photocrosslinked for about 30 seconds; and empty RS (G3) that did notreceive any treatment.

FIG. 1B is a schematic diagram illustrating that the 3 groups of FIG. 1Awere cultured in osteogenic media (OM) and implanted subcutaneously.

FIG. 1C is a schematic timeline of the processes of FIGS. 1A and 1B.

FIG. 2A shows a sectioned G1 construct after about 13 day in vitroculture.

FIG. 2B shows an enlarged view of the boxed area of FIG. 2A.

FIG. 2C shows a polarized light microscopy (Pol) image of FIG. 2B.

FIG. 2D shows a double immunofluorescent (IF) image of the G1 constructof FIG. 2A.

FIG. 2E shows a sectioned G1 construct after about 4 weeks in vivoculture.

FIG. 2F shows an enlarged view of the boxed area of FIG. 2E.

FIG. 2G shows a Pol image of FIG. 2F.

FIG. 2H shows a double IF image of the G1 construct of FIG. 2E.

FIG. 2I shows a sectioned G1 construct after about 8 weeks in vivoculture.

FIG. 2J shows an enlarged view of the boxed area of FIG. 2I.

FIG. 2K shows a Pol image of FIG. 2J.

FIG. 2L shows a double IF image of the G1 construct of FIG. 2I.

FIG. 3A shows a sectioned G2 construct after about 13 days in vitroculture.

FIG. 3B shows an enlarged view of the boxed area of FIG. 3A.

FIG. 3C shows a Pol image of FIG. 3B.

FIG. 3D shows a sectioned G2 construct after about 4 weeks in vivoculture.

FIG. 3E shows an enlarged view of the boxed area of FIG. 3D.

FIG. 3F shows a Pol image of FIG. 3E.

FIG. 3G shows a double IF image of the G2 construct of FIG. 3D.

FIG. 3H shows a sectioned G2 construct after about 8 weeks in vivoculture.

FIG. 3I shows an enlarged view of the boxed area of FIG. 3H.

FIG. 3J shows a Pol image of FIG. 3I.

FIG. 3K shows a double IF image of the G2 construct of FIG. 3H.

FIG. 4A shows a sectioned G3 construct after about 4 weeks in vivoculture.

FIG. 4B shows an enlarged view of the boxed area of FIG. 4A.

FIG. 4C shows a Pol image of FIG. 4B.

FIG. 4D shows a double IF image of the G3 construct of FIG. 4A.

FIG. 4E shows a sectioned G3 construct after about 8 weeks in vivoculture.

FIG. 4F shows an enlarged view of the boxed area of FIG. 4E.

FIG. 4G shows a Pol image of FIG. 4F.

FIG. 4H shows a double IF image of the G3 of FIG. 4E.

FIG. 5A shows Rubust rh-Mitochondria staining of GelMA encapsulatedhDPSCs and HUVECs at about 13 days in vitro.

FIG. 5B illustrates that DPSCs and HUVECs in about 4 weeks in vivoimplanted G1 constructs.

FIG. 5C illustrates a weak signal in about 8 weeks in vivo implantedconstructs.

FIG. 5D illustrates rh-Mitochondria antibody identified human DPSC andHUVECs in about 4 weeks in vivo acellular GelMA implants.

FIG. 5E illustrates rh-Mitochondria antibody identified human DPSC andHUVECs in about 8 weeks in vivo acellular GelMA implants.

FIG. 5F illustrates a human gingiva positive control for therh-Mitochondria antibody.

FIG. 5G illustrates about 4 weeks in vivo empty RS implants.

FIG. 5H illustrates about 8 weeks in vivo empty RS implants.

DETAILED DESCRIPTION

While the inventions described herein are susceptible of embodiment inmany different forms, there is shown in the drawings and will herein bedescribed in detail preferred embodiments of the inventions with theunderstanding that the present disclosure is to be considered as anexemplification of the principles of the inventions and is not intendedto limit the broad aspects of the inventions to the embodimentsillustrated.

The embodiments described herein are directed to compositions andmethods for regenerating pulp-like tissues in tooth root segments (RS)injected with human dental pulp stem cells (hDPSCs) and human umbilicalvein endothelial cells (HUVECs) and/or vascular endothelial growthfactor (VEGF) encapsulated in (e.g., 5% (w/v)) gelatin methacrylate(GelMA) hydrogel. It is contemplated that the GelMA may present at anysuitable concentration including, but not limited to, about 3% to about5% (w/v).

hDPSC may be isolated from dental pulp. In some embodiments, the dentalpulp is from a mature tooth. The mature tooth may be a wisdom tooth, acryopreserved tooth, or any other suitable mature tooth. In otherembodiments, bleeding may be induced at or near the apex of the tooth toobtain hDPSCs from the blood.

According to one method, a dental tissue regenerative composition isformed. hDPSCs, at least one of HUVECs or VEGF, a GelMA hydrogel, and aphoto-initiator are combined. The GelMA hydrogel is cross-linked withthe hDPSCs and the at least one of HUVECs or VEGF by exposure to a UV orvisible light.

According to another method, pulp-like tissues in a tooth root segmentof a tooth are regenerated by injecting GelMA hydrogel and aphoto-initiator into a tooth root segment. The GelMA hydrogel and thephoto-initiator are combined with a mixture of (1) hDPSC and (2) atleast one of HUVECs or VEGF, either before or after injection into thetooth root segment. The GelMA hydrogel is cross-linked with the mixtureby exposing the composition to a UV or visible light. The cross-linkingmay be performed either prior to or after injection in the tooth rootsegment.

The duration of the exposure to the UV or visible light may range fromabout 15 seconds to about 35 seconds. In another embodiment, theexposure ranges from about 18 seconds to about 30 seconds.

To demonstrate the reliability of the compositions described herein, RSinjected with acellular GelMA alone and empty RS were used as controlsand compared with RS injected with hDPSCs and HUVECs encapsulated inabout 5% GelMA hydrogel. Combined hDPSCs and HUVECs (in a ratio of about1:1) were encapsulated in about 5% GelMA and injected into a RS orificeof about 6 mm length and about 2-3 mm wide. White mineral trioxideaggregate was used to seal one of the orifices while the other was leftopen. Samples were cultured in vitro in osteogenic media (OM) for about13 days and subsequently implanted subcutaneously in nude rats for about4 weeks and about 8 weeks. At least five sample replicates were used foreach experimental group. The methods are described in more detail below.

Materials and Methods

Human Teeth Procurement, Dental Cell Isolation, and in Vitro Expansion

Human teeth extracted for clinically relevant reasons were obtained fromthe Tufts University School of Dental Medicine and the Back Bay OralMaxillofacial Clinic in Boston, Mass. hDPSCs were isolated from dentalpulp obtained from extracted wisdom teeth. HUVECs (PSC100010, ATCC,Manassas, Va.) were pre-cultured in vascular basal media (VBM)(PCS100030, ATCC) with vascular endothelial growth factor (VEGF) kit(PCS10004, ATCC) and humidified in about 5% CO₂ at about 37° C. Expandedcells were cryopreserved in about 10% dimethyl sulfoxide (DMSO) inappropriate culture media until use.

Cryopreserved Passage 2 hDPSCs were cultured in OM, including Dulbecco'sModified Eagle's medium (DMEM)/F12 supplemented with about 1% PSA, about10% FBS, about 100 nM dexamethasone, about 10 mM beta glycerolphosphate, and about 0.05 mM ascorbic acid for about 13 days.Cryopreserved Passage 5 HUVECs were expanded in vitro in VBM with mediachanges about every 2 days.

Research Design

Three groups of root segments (RS) were examined in this study: (1)G1-GelMA encapsulated hDPSC/HUVEC filled RS; (2) G2-acellular GelMAfilled RS; and (3) G3 -empty RS. Replicate samples were cultured forabout 13 days in OM in vitro. Five replicates of each group were fixedand analyzed using histological and immunohistochemical (IHC) methods.The remaining RSs were implanted subcutaneously in nude rats and grownfor about 4 weeks and about 8 weeks (see FIGS. 1A-1C).

FIGS. 1A-1C illustrate a schematic of the design according to oneembodiment. Specifically, GelMA encapsulated cell constructs (G1)consisting of cultured hDPSCs and HUVECs that were encapsulated in about5% of GelMA, injected into human RS, and photocrosslinked for about 30seconds. Acellular GelMA RSs (G2) were also photocrosslinked for about30 seconds. Empty RS (G3) did not receive any treatment. FIG. 1B showsthe 3 groups of FIG. 1A being cultured in OM for about 13 days and thenimplanted subcutaneously for about 4 weeks or about 8 weeks. FIG. 1Cillustrates a schematic timeline of the processes of FIGS. 1A, 1B.

GelMA Preparation

Lyophilized GelMA was fully dissolved in DMEM/F12 media, and about 0.1%(w/v) of photo-initiator (PI) (Irgacure2959, Sigma, St. Louis, Mo.) wasadded to create about 5% GelMA solution (denoted as 5% GelMA), which wassterilized by filtration using a 0.22 μm filter and stored in the darkuntil use.

It is contemplated that the photo-initiator may be present at anysuitable concentration including, but not limited to, about 0.05% toabout 0.5% (w/v). In some embodiments, the concentration of thephoto-initiator is about 0.1% (w/v). It is also contemplated that anysuitable photo-initiator or combination of photo-initiators may be used.For example, the photo-initiator may include1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy methyl-1-propane-1-one,azobisisobutyronitrile, benzoyl peroxide, di-tert-butyl peroxide,2,2-dimethoxy-2-phenylacetophenone, Eosin Y, or any combination thereof

Tooth Root Section Selection Criteria, Disinfection, and MTA Placement

Teeth were collected from healthy patients aged about 15 to about 30years old, including single and multi-rooted teeth with Type I & VVertucci root canal configurations. Teeth containing caries, Type II-IVVertucci root canal configuration, calcified canals, or those with priorroot canal treatment were excluded. Tooth RSs were cut using sterilized330 and fissure burs, from the coronal and middle third of the roots tominimize root curvature. RS were about 6 mm in length and of about 2-3mm orifice width to facilitate injection of cellular and acellularGelMA. The pulpal space lumen was enlarged using Gates Glidden (GG)sizes 1 and 2 and the previously mentioned burs. Next, RSs were preparedusing ethylenediaminetetraacetic acid (EDTA), Sodium hypochlorite(NaOCL), and phosphate buffered saline (PBS) washes. To test for anyremaining microbial contamination, RS were cultured in DMEM/F12 media atabout 37° C. for about 4 days. White Mineral Trioxide Aggregate (WMTA,ProRoot DENTSPLY Tulsa Dental Specialties, Tulsa, Okla.) was then usedto create a plug at one side of each RS to mimic the clinical situation,while the other end was left open to allow host cell invasion.

Cell Preparation and GelMA Encapsulation

Confluent flasks of hDPSCs and HUVECs were trypsinized and resuspendedin their respective media, and cell densities were calculated using aCountess Automated Cell Counter (Invitrogen™, Carlsbad, Calif.). A totalof about 6×10⁵ hDPSCs and about 6×10⁵ HUVECs (in a ratio of about 1:1)were combined into one 50 ml tube and resuspended in about 0.5 mL offiltered about 5% GelMA. Approximately 30 μL of GelMA with hDPSCs andHUVECs (about 7.0×10⁴ cells), or acellular GelMA, were injected into RSsand photocrosslinked via exposure to about 9.16 W/cm² UV light for about20 seconds using an Omnicure 52000 (Lumen Dynamics Group Inc.,Mississauga, ON, Canada).

Subcutaneous Implantation in Nude Rats

Using Tufts University approved IACUC Protocols, implants were placed in8 female Nude rats aged about 4 weeks to about 6 weeks. Four incisionswere created, two on each side, and fascia was separated from the muscleto form a lateral sac deep enough to hold an individual RS. Afterimplantation, the incisions were closed with wound clips. The rats werechecked every day for one week, and Buprenorphine was administered onceevery two days for one week.

Root Segment (RS) Harvest

Replicate RSs were harvested at about 4 weeks and about 8 weeks usingTUSDM approved IACUC protocols, washed 3 times in PBS, fixed in about10% Formalin overnight, washed with PBS, and decalcified in about 10%EDTA at a pH of about 7 for about 4 months. Decalcification wasmonitored by taking about 5 ml of the about 10% EDTA solution each weekand adding a drop of about 1.0 M HCL plus about 1 ml of saturatedAmmonium Oxalate. The solution was mixed thoroughly, allowed to sit forabout 20 minutes, and monitored for CaPO₄ precipitate. Lack ofprecipitate formation was defined as substantially completedecalcification.

Cryosectioning

Samples were prepared for cryostat sectioning as previously described.Samples were wrapped in plastic wrap to prevent dehydration and storedat a temperature of about-80° C. A cryostat (Leica Biosystems, Nussloch,Germany) set at about −21° C. was used to section samples at about 10 μmor about 30 μm intervals, for histological/IF and confocal analyses,respectively. Magic Tape (Cryofilm Type2C, Section-Lab, Hiroshima,Japan) was used to transfer sections to Superfrost® Plus MicroscopeSlides Precleaned (Fisher Scientific, Atlanta, Ga.) and stored at about−20° C. until use.

Hematoxylin and Eosin (H&E) Stain

To avoid dislodging samples from Magic-Tape glass slides, the slideswere carefully dipped three times in deionized water (DI H₂O) andstained with H&E stain using standard protocol. Dehydration wasperformed by dipping two or three times in about 95% ethanol (EtOH) andabout 100% EtOH and one time in Xylene. Samples were cover slipped usingPermount (Fisher Scientific, Atlanta, Ga.).

Double Immunofluorescent (IF) Histochemical Analyses

IF was performed as briefly described above. Primary antibodies includedmouse αCD31 (1:200, ab187377, Abcam, Cambridge, Mass.) and rabbit αVimentin (1:25, bs-0756R, Bioss, Woburn, Mass.). Secondary antibodiesincluded goat a mouse (Invitrogen, 568, 1:50, West Grove, Pa.) and goatα Rabbit (Invitrogen, 488, 1:50, West Grove, Pa.). Mouse αrh-Mitochondria (1:25, Millipore Sigma, MAB1273, Temecula, Calif.) wasused to discriminate between HUVEC and host endothelial cells.

Results

Analyses of harvested samples found that pulp-like tissues formed inhDPSC/HUVEC encapsulated GelMA-filled RS and that host cell infiltrationwas observed in the acellular GelMA and empty RS groups. hDPSCs andHUVECs were identified using IF histochemical analysis for Vimentin andCD31, respectively. Construct cells were distinguished from host cellsusing IF with an anti-rh-mitochondrial antibody.

(G1) GelMA-Encapsulated Cell Tooth Root Segments

Prior to encapsulation, in vitro cultured hDPSCs and HUVEC exhibitedtypical fibroblastic and cobblestone-like morphologies, respectively(not shown). G1 (GelMA encapsulated hDPSC/HUVEC RSs) exhibitedcellularized bioengineered pulp-like tissue after about 13 days in vitroculture (see FIGS. 2A, 2B). Cellularity of the G1 constructs appeared toincrease in about 4 week and about 8 week in vivo implanted constructs(see FIGS. 2E, 2F, 2I, 2J), to eventually occupy substantially all ofthe pulpal space not containing MTA. Polarized light imaging revealedcollagen deposition 102, 104 in about 4 week and about 8 week in vivoimplanted G1 samples. Collagen deposition 102 appeared more organized atabout 8 weeks as compared to collagen deposition 104 at about 4 weeks(see FIGS. 2K and 2G). Neovascularization 106a, 106b was observed inabout 4 week in vivo implanted samples (FIG. 2F). The 8 week G1 in vivoimplants showed patent blood vessels containing red blood cells 108(FIG. 2J). IF analyses of VM expressing 110 hDPSCs and CD31 expressing112 HUVECs in G1 RSs revealed neovascular network formation overimplantation time (see FIGS. 2D, 2H, 2L).

As briefly discussed above, FIGS. 2A-2L show GelMA encapsulatedhDPSC/HUVEC RSs of G1. FIG. 2A shows a sectioned G1 construct afterabout 13 days in vitro culture. FIG. 2A shows bioengineered pulp and thelocation of MTA plug. FIG. 2B is an enlarged view of the boxed area 116of FIG. 2A showing cellularity of construct and undegraded GelMA 118. Asshown in FIG. 2C, polarized light (Pol) microscopy showed no orsubstantially no ECM deposition. FIG. 2D shows a double IF of CD31expressing HUVECs 112 and VM expressing hDPSCs 110.

As shown in FIG. 2E, a sectioned 4 week in vivo implanted constructexhibited higher cell density and organization as compared to 13 days invitro (see FIG. 2A). As shown in FIG. 2F, odontoblast-like cells 106awere present at the pulp-dentin interface, and functionalneovascularization-containing red blood cells 106b were observed. Asseen in FIG. 2G, Pol microscopy showed obvious ECM deposition 104 atabout 4 weeks as compared to about 13 day in vitro cultured RS (see FIG.2C). As shown in the double IF image of FIG. 2H, there was increasedcellularity of both hDPSCs 110 and HUVECS 112.

FIG. 2I shows that about 8 week in vivo implanted G1 RSs exhibited highcellularity. As shown in FIG. 2J, implant vascularization 108 appearedmore distinct as compared to about 4 week G1 implants (see FIG. 2F). Asseen in FIG. 2K, increased collagen deposition 102 was apparent at about8 weeks in vivo as compared to about 4 week in vivo implanted G1 samples(see FIG. 2G). Double IF revealed high cellularity of hDPSCs and HUVECS.The scale bars of FIGS. 2A-2K were as follows: FIGS. 2A, 2E, 2I=500 μm;FIGS. 2B, 2C, 2F, 2G, 2J, 2K=20 μm; and FIGS. 2D, 2H, 2L=50 μm. 127×70mm (300×300 DPI).

(G2) Acellular GelMA-Filled Tooth Root Segments

G2, Acellular GelMA RSs were found to exhibit degraded GelMA after about13 day in vitro culture (see FIGS. 3A, B). FIG. 3A shows that sectioned13 day in vitro cultures of acellular RS exhibited degraded GelMA andremnant MTA. FIG. 3B is an enlarged view of the boxed area 122 of FIG.3A and shows remnant GelMA. As shown in FIG. 3C, polarized light imagingdid not detect organized collagen. In about 4 week (FIGS. 3D, 3E) andabout 8 week (FIGS. 3H, 3I) in vivo implanted G2 constructs, host cellswere present on both the GelMA and dentin surfaces, and host celldensity appeared to increase over in vivo implantation time. As shown inFIG. 3D, about 4 week in vivo G2 implants exhibited increasedcellularity as compared to 13 days in vitro cultured G2 constructs (seeFIG. 3A). The area 124 in FIG. 3D (enlarged in FIG. 3E) shows hostcellularity, attachment to GelMA, and vascularity including host redblood cells 126.

As shown in FIG. 3H, sectioned about 8 week in vivo implants revealedhigh cellularity and invading host cells. The boxed area 128 in FIG. 3H(enlarged in FIG. 3I) revealed generally undegraded GelMA that persistedin about 8 week in vivo implanted G2 constructs and host cells 130.Generally undegraded GelMA was detectable in both about 4 week (FIG. 3E)and about 8 week (FIG. 3I) in vivo implanted constructs. Red blood cells126, 130 were observed in acellular GelMA RS (see FIGS. 3E and 3I).Polarized light microscopy revealed host cell collagen deposition 132 inabout 4 week and about 8 week in vivo implanted G2 constructs (see FIG.3F, 3J). As shown in FIG. 3F, polarized light revealed host cell derivedECM 132a. As shown in FIG. 3J, polarized light microscopy revealedincreased host derived ECM deposition 132b as compared to about 4 weekin vivo implanted G2 constructs (FIG. 4F).

Double IF revealed VM-expressing host mesenchymal stem cells (MSCs) 136and CD31-expressing host endothelial cells 138 in about 4 week (FIG. 3G)and 8 week (FIG. 3K) in vivo implanted G2 constructs. As shown in FIG.3G, double IF revealed host CD31-expressing endothelial 138a andVimentin-expressing mesenchymal cells 136a. As shown in FIG. 3K, doubleIF revealed CD31-expressing host endothelial cells 138b andVM-expressing host mesenchymal cells 136b in about 8 week in vivoimplanted G2 constructs. The scale bars used in FIGS. 3A-3K are asfollows: FIGS. 3A, 3D, 3H=500 μm; FIGS. 3B, 3C, 3E, 3F, 3I, 3J=20 μm;and FIGS. 3G, 3K=50 μm. 127×70 mm (300×300 DPI).

(G3) Empty Tooth Root Segments

In vivo implanted G3, empty RSs, also exhibited host cell infiltrationat about 4 weeks and about 8 weeks (FIGS. 4A, 4B and FIGS. 4E, 4F,respectively). A shown in FIG. 4A, G3 constructs at about 4 weeks invivo implantation showed host encaps6lation and cellularity within thepulpal space. As shown in FIG. 4B (the enlarged view of boxed area 148of FIG. 4A), host cells attached to the dentin surface, and host cellsinfiltrated into the pulpal space.

As shown in FIG. 4E, about 8 week in vivo implanted G3 constructsexhibited increased cellularity as compared to about 4 week G3 implants(see FIG. 4A). As shown in FIG. 4F, more distinct host cellularity wasobserved in about 8 week as compared to about 4 week in vivo implantedG3 samples (see FIG. 4B). Host cell-derived ECM in implanted G3constructs appeared more organized at about 8 weeks (see FIG. 4G) ascompared to about 4 weeks (see FIG. 4C), as revealed by polarized lightmicroscopy. As shown in FIG. 4G, polarized light imaging revealedincreased host ECM deposition 154 at about 8 weeks as compared to about4 weeks (see FIG. 4C) in vivo implantation. As shown in FIG. 4C,polarized light revealed dentin ECM 152 only. IF histochemical analysesrevealed CD31 positive host vascularity 158 and VM-expressing MSCs 156in in vivo implanted empty RSs (see FIGS. 4D, 4H). As shown in FIG. 4D,double IF revealed the presence of host endothelial and mesenchymaltissues. Double IF showed similar results in the about 8 week in vivoimplanted G3 constructs (FIG. 4H) as the about 4 week in vivo implantedG3 constructs (FIG. 4D). Neovasculature of empty RSs appeared lessorganized than that present in acellular GelMA RSs and much less thanwas observed in in vivo implanted hDPSC/HUVEC encapsulated GelMA RSs.The scale bars of FIGS. 4A-4H are as follows: FIGS. 4A, 4E=500 μm; FIGS.4B, 4C, 4F, 4G=20 μm; and FIGS. 4D, 4H=50 μm. 127×48 mm (300×300 DPI).

Discriminating Between Human and Host Cells

To evaluate the long-term survival of GelMA-encapsulated hDPSC and HUVECcells and to discriminate between human and host cells, sectionedsamples were examined using an anti-rh-mitochondria antibody thatrecognizes human cells and does not cross-react with rat cells. Humancells 160 were identifiable in G1 constructs after about 13 days invitro culture and after about 4 weeks in vivo implantation (see FIGS.5A, 5B) but not at about 8 weeks (see FIG. 5C) in vivo. In FIG. 5A,rubust rh-Mitochondria staining of GelMA encapsulated hDPSCs and HUVECsat 13 days in vitro is shown. As shown in FIG. 5B, human DPSCs andHUVECs were also clearly detected in about 4 week in vivo implanted G1constructs. As shown in FIG. 5C, weak signal in about 8 week implantedcontructs indicated that implanted human cells were replaced by hostcells. Acellular GelMA and empty RSs generally exhibited no positiverh-mitochondria expression (see FIGS. 5D, 5E, 5G, 5H). Positive controlhuman gingiva was positive for rh-mitochondria (see FIG. 5F). As shownin FIGS. 5D, 5E, rh-Mitochondria antibody identified human DPSC andHUVECs in about 4 week and about 8 week in vivo acellular GelMAimplants. As shown in FIGS. 5G and 5H, about 4 week and about 8 week invivo empty RS implants exhibited similar results as the acellular GelMAgroup of FIGS. 5D, 5E. Namely, both groups exhibited host cellinfiltration and no rh-Mitochondrial marker expression. FIG. 5F showshuman gingiva positive control for the rh-Mitochondria antibody. Thescale bars of FIGS. 5A-5H are as follows: 50 μm. 127×95 mm (300×300DPI).

DISCUSSION

One of the goals of the embodiments described herein was to define amore effective, clinically relevant method for pulpal revascularizationand regeneration in human tooth root segments. First, hDPSCs were usedin combination with HUVECs, based on the importance of both cell typesfor pulpal tissue formation and vascularity and on the successful use ofHUVEC-derived neovasculature to facilitate implant viability andintegration with host vasculature in in vivo implants. Moreover, it wasreported that DPSCs contributed to increased neovascular networkformation by facilitating HUVEC migration and by increasing vascularendothelial growth factor (VEGF) expression.

As discussed above, hDPSCs and HUVECs (and/or, in some embodiments,vascular endothelial growth factor (VEGF)) were encapsulated in GelMA, apreferred hydrogel scaffold for 3D tissue engineering applications basedon its ability to facilitate cell attachment, spreading, proliferation,and promotion of host cell interactions. The ability to easily injectand photo-crosslink GelMA-encapsulated cells also makes GelMA anattractive scaffold for clinically relevant pulpal regenerationprocedures. It is contemplated, however, that other hydrogel scaffoldsmay also or alternatively be used.

It is contemplated that the compositions described herein may beinjected into a tooth root segment using any suitable device or method.In one non-limiting embodiment, a double barrel syringe may be used, inwhich the first barrel may include a photo-initiator and the secondbarrel may include GelMA. The contents of the first and second barrelsmay be simultaneously injected into the tooth root segment, or they maybe injected in sequence.

Dental and endothelial cell-encapsulated GelMA may be used as constructsfor 3D biomimetic tooth bud models. In such embodiments, GelMA formulasgenerally support DE and DM cell attachment, spreading, metabolicactivity, and neo-vasculature formation by co-seeded endothelial cells(HUVECs). Selected GelMA formulas were used to create 3D tooth budsconsisting of a biomimetic enamel organ layer (DE and HUVECsencapsulated in about 3% GelMA) and a biomimetic pulp organ (DM andHUVECs encapsulated in about 5% GelMA). The resultant 3D biomimetictooth bud generally supported dental cell differentiation,vascularization, and in vivo formation of mineralized osteodentintissues of specified size and shape. In the embodiments describedherein, GelMA is used to create a biomimetic pulp organ containing bothhDPSC and HUVECs encapsulated in about 5% GelMA, created in human toothRSs, to study pulp regeneration.

H&E staining and polarized light (Pol) microscopy revealed that GelMAencapsulated hDPSC and HUVECs contributed to the formation ofbioengineered pulp-like tissue that exhibited increased cellularity overin vivo implantation time (see FIG. 2 ). These data also showed thatGelMA scaffold in G1 (cell-seeded) constructs had largely degraded afterabout 8 week in vivo implantation but was still detectable in G2(acellular GelMA RSs) after about 8 weeks. Tight association of hosttissues to the GelMA scaffold (which is generally indicative of goodbiocompatibility) and over the inner dentin surface of implanted RSs wasobserved (see FIGS. 3, 4 ). Additionally, acellular GelMA appeared topromote host tissue infiltration, proliferation, and vascularization ofthe implant (see FIG. 3 ).

Within each RS, WMTA was used to seal off one end of each RS to mimicclinical treatment of a natural tooth. Remnant MTA is clearlyidentifiable in some sectioned implants, although it appeared to havebeen lost from others during sample processing. Reparative dentinformation below the MTA was generally not observed, possibly due to therelatively short duration (1-2 months).

As discussed above, GelMA supported dental and HUVEC cell proliferation.It is noteworthy that host cell-derived ECM elaborated in G2 (acellularRSs) appeared somewhat more organized than that formed in G3 (empty RSs)after about 4 weeks and about 8 weeks in vivo implantation. Thus, GelMAmay facilitate host cell infiltration and organization prior todegradation.

As described above, a functional vascularized network is required forthe long term survival of bioengineered tissues and for properintegration with the recipient host. In natural tissues, blood vesselsare composed of a luminal endothelial cell layer, surrounded by a layerof smooth muscle cells. Mesenchymal stem cells (MSCs) and endothelialcells have been shown to exhibit the ability to self-organize intocapillary-like networks after encapsulation in GelMA hydrogel in vitroand in vivo. Confocal analyses and immunofluorescent (IF) histochemicalanalyses were used in the embodiments discussed herein to examineneo-vessel formation and organization within in vitro cultured and invivo implanted samples. Elaborate neo-vascular and capillary-likenetwork formation were identified in all in vivo implanted groups afterabout 4 weeks and about 8 weeks. However, more organized neovasculaturenetworks were observed in G1 (GelMA-encapsulated hDPSC/HUVEC RSs) ascompared to acellular and empty RSs. Importantly, the presence of hostred blood cells within the bioengineered vasculature confirmed theability of G1 constructs to form functional vascular networks in vivo,as would be required to support tooth integration and growth afterimplantation. IF analyses using rh-mitochondria showed that the GelMAencapsulated hDPSCs and HUVECs survived for about 4 weeks in vivoimplantation, but were generally not detectable at about 8 weeks in vivo(see FIG. 5C).

In conclusion, GelMA hydrogels may be used to support the formation ofhDPSC/HUVEC derived, highly cellularized and vascularized pulp-liketissue formation in human tooth root segments and, to a lesser degree,in acellular GelMA constructs. GelMA may also support hDPSC/HUVEC cellattachment and proliferation and attachment for host cells. Cell-seededGelMA hydrogels promote the establishment of host vasculature within thesegments and promote extracellular matrix (ECM) deposition. Theseresults validate GelMA encapsulated human cell constructs as a promisingalternative therapy for clinically relevant pulpal revascularization, toreplace infected pulpal tissues in pulpotomy procedures, and/or as atemporary intracanal medicament.

Although the embodiments described herein generally include humanumbilical vein endothelial cells (HUVECs), it is contemplated thatvascular endothelial growth factor (VEGV) may be used instead of or inaddition to the HUVECs.

While the embodiments herein have been described with reference to oneor more particular embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. Each of these embodiments andobvious variations thereof is contemplated as falling within the spiritand scope of the invention. It is also contemplated that additionalembodiments according to aspects of the present invention may combineany number of features from any of the embodiments described herein.

1. A dental tissue regenerative composition comprising a combination of(1) human dental pulp stem cells and (2) at least one of human umbilicalvein endothelial cells or vascular endothelial growth factor, whereinthe combination is encapsulated in a light-activated gelatinmethacrylate hydrogel. 2-35. (canceled)